Biological and molecular characteristics of four potato virus Y (PVY) isolates based on partial coat protein gene

Document Type : Research Paper

Authors

• Prastoo Poursharifi ¹
• Akbar Dizadji ²
• Parisa sharifinezamabad ³
• Mina Koohi Habibi ⁴
• Hadi Khateri ⁵

¹ Department of Plant Protection, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran

² Department of Plant Protection, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran.

³ Department of Plant Protection, Agricultural and Natural Resources and Research Center of Golestan province, Gorgan, Iran.

⁴ Department of Herbal Medicine, Faculty of Agriculture, University of Tehran. Karaj. Iran

⁵ Department of Plant Protection, College of Agriculture, Razi University, Kermanshah, Iran

Abstract

Keywords

• Phylogeny
• strain
• serology
• Physalis

Extended Abstract

Introduction

Potato virus Y (PVY, genus Potyvirus, family Potyviridae), one of the top 10 most important plant viruses (Scholthof et al., 2011), is a serious problem in solanaceous crops worldwide (Green et al., 2017a; Kerlan, 2006; Quenouille et al., 2013). Currently, five non-recombinant (PVY^O, PVY^Eu-N, PVY^NA-N, PVY^C, and PVY^O-O5) and nine recombinant strains (PVY^N:O, PVY^N-Wi, PVY^NTNa, PVY^NTNb, PVY-^NE11, PVY^E, and PVY^-SYR-I, ^-II, and ^-III) have been defined (Galvino-Costa et al., 2012; Green et al., 2017b; Karasev & Gray, 2013). Molecular studies are useful tools to shed light on the molecular basis of virus geographical spread and adaptation to new hosts and for designing appropriate epidemic control strategies (Elena et al., 2011; Jones, 2009). PVY has been reported in several regions of Iran (Hosseini et al., 2011; Pourrahim & Farzadfar, 2016; Pourrahim et al., 2007; Sadeghi et al., 2008; Shamsaddin Saeed et al., 2008; Toosi et al., 2004). In this study, biological, serological and molecular analysis of four PVY isolates was performed based on the C-terminal of the coat protein genomic region.

Materials and Methods

A total of 1178 symptomatic leaf samples of pepper, potato, tobacco and physalis were collected from the fields in Karaj, Urmia, Sanandaj, Shiraz, Qazvin, Varamin and Hamedan counties during 2010-2011. The samples were tested serologically by double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) as described by Clark and Adams (1977) using PVY-specific polyclonal antibodies (DSMZ, AS-0137).

Four ELISA-positive samples were biologically purified by serial single local lesion transfers and propagated on Nicotiana glutinosa, then mechanically inoculated on a number of indicator plants. Total RNA was extracted from infected N. glutinosa leaves by RNeasy Plant Mini Kit (Qiagen, Hilden Germany) and subjected to RT-PCR using CPUTR-F/CPUTR-R primer pair to amplify the virus genomic 3' end (Bukovinszki et al., 2007). Amplified fragments were extracted from the gel using a GF-1 Gel DNA recovery kit (Vivantis, Malaysia) then sequenced directly in both directions (Macrogen Inc., South Korea).

Immunocapture-RT-PCR (IC-RT-PCR) was performed by using strain-specific primers as described by Boonham et al. (2002).

The nucleotide sequences of Iranian isolates were compared against the online NCBI nt database using BLASTn, then aligned to a set of PVY sequences available in GenBank. Pairwise nucleotide identities  were obtained by SDT v. 1.2 (Muhire et al., 2014), phylogenetic tree reconstruction was done by MEGA X (Kumar et al., 2018).

Results and Discussion

PVY was detected by DAS-ELISA in samples of tobacco (27.27 %), potato (20.4 %) and physalis (18.75 %). Interestingly, although a large number of pepper samples were tested, none of them were infected by PVY. Four biologically purified PVY isolates (Po2, Po5, Tob2 and Ph1), obtained from potato, tobacco and physalis caused local lesions on Chenopodium amaranticolor and systemic symptoms on Capsicum annuum, Datura metel, Solanum lycopersicum, Nicotiana debneyi, N. glutinosa and N. rustica, with minor differences in experimental host range. RT-PCR resulted in the amplification of the 3´ genomic end of PVY isolates with the expected size of 1200 bp. Phylogenetic analysis based on nucleotide sequences of partial coat protein gene (encoding N- terminal region of coat protein) of four PVY Po2, Ph1, Po5 and Tob2 isolates in this study (with NCBI GenBank accession numbers of PP195788, PP195789, PP195790 and PP195791, respectively) with other PVY sequences available in GenBank revealed that Ph1, Po5 and Tob2 isolates grouped into PVY^N/NTN clade, while only Po2 grouped into PVY^O clade. Pairwise nucleotide sequence comparisons of 55 PVY genomes (for 51 isolates previously reported and the 4 isolates reported in this study) using SDT revealed that Tob2, Po5 and Ph1 isolates have >94- 100% and Po2 has >99% identity with PVY^N/NTN and PVY^O isolates, respectively. Immunocapture-RT-PCR (IC-RT-PCR) by using the strain-specific primers resulted in the amplification of a cDNA fragment with the expected size of 545 bp in Tob2, Po5 and Ph1 isolates and of 609 bp in Po2 one, which confirmed the phylogenetic results. No recombination event was detected in the partial cp gene between 55 PVY isolates.

The information obtained can be a key element in future research to develop improved control strategies for potato virus diseases in Iran.

Conclusion

The present study showed the occurrence of PVY^N/NTN and PVY^O in different hosts based on biological characteristics and molecular analyses of the partial cp gene nucleotide sequence. To our knowledge, this is the first report of the occurrence of PVY^N/NTN on Physalis divaricata, while whole genome sequencing of this isolate is needed.